The Proteasome
The proteasome is a large, multi-protein complex present in both the cytoplasm and the nucleus of all eukaryotic cells. The 26s proteasome is involved in the constitutive and controlled degradation of proteins that control vital processes such as cell cycle progression, differentiation, and apoptosis. It also participates in the clearance of misfolded and damaged proteins and in the generation of peptides for MHC class I restricted antigen presentation (Schwarz et al, Annual Review of Medicine. 50:57–74 (1999). Recently, inhibition of the activity of the proteasome was observed in cells expressing aggregation-prone proteins, which are at the center of several neurodegenerative diseases (Bence et al. Science. 2001 May 25;292(5521):1552–5).
The diverse roles of proteasomal degradation place the proteasome at the core of pathological processes such as inflammation, autoimmunity, neurodegenerative diseases, and cancer. This key position in important pathological systems has motivated efforts to elucidate the mechanisms of action of the proteasome and to identify compounds to modulate its activity. Several inhibitors of the proteasome are now tested in clinical trials for cancer therapy (Adams, Trends Mol Med. 2002;8(4 Suppl):S49–54).
Proteins are identified for degradation by the proteasome via covalent modification with the small polypeptide ubiquitin or by sequence motifs in the target protein, which act as proteolytic signals (Schwarz Annual Review of Medicine 50:57–74,1999). One such motif, the PEST sequence, is found extensively in short-lived proteins (Rogers et al Science. 234(4774):364–8 1986). Mouse Ornithine DeCarboxylase (MODC) is one of the shortest half-lived proteins in mammals. The constitutive degradation of MODC by the proteasome is controlled by PEST sequences in its carboxy terminus (Loetscher et al. J Biol. Chem. 1991 Jun. 15;266(17):11213–20; Ghoda et al Mol Cell Biol. 1992 May;12(5):2178–85).
Proteomics
The study of proteasome function is also an important consideration in the emerging field of proteomics, the study of the proteins that are the gene products. A broad definition of proteomics is the effort to establish the identities, quantities, structures, and biochemical and cellular functions of all proteins in an organism, organ, or organelle, and how these properties vary in space, time, or physiological state.
As an example of immediate goals of the proteomics area of research is the Human Proteomics Initiative, which has as it goal annotating each know protein, providing information that includes the description of protein function, domain structure, subcellular location, post-translational modifications, splice variant, and similarities to other mammalian proteins.
A core need of proteomics efforts is to understand the cells specific protein degradation processes as these effect the levels and identities of proteins in the cell. Cellular levels of proteins will often vary substantially from the initial levels of productions due to degradation. Having a sensitive means of assessing the level of proteasome function in response to various factors would be of great value to this field of research.
Reporter Constructs
In monitoring biological systems, a number of reporter molecules have been usefully employed in tracking the cellular production and ultimate fate of a protein of interest by linking its genetic code to that of the reporter molecule. Then, when the reporter molecule is detected in the cell, this result indicates that the protein of interest is also present or not present. These reporters can include such molecules as luciferases among other reporters. However, the most widely used of these molecules are the fluorescent proteins, typically derived from marine animals, because currently they are the main source of non-invasively assessing the biological systems.
Fluorescent proteins are widely used as reporters for the detection of events in live cells. After the Green Fluorescent Protein (GFP and the enhanced EGFP) and its yellow (YFP) and cyan (CFP) variants, the discovery of the reef coral fluorescent proteins (RCFPs) ZsGreen, ZsYellow, AmCyan, AsRed (red), DsRed (orange-red) and HcRed (far-red) has expanded the spectrum of colors available for these studies (Matz et al., Nat Biotechnol. 1999 October;17(10):969–73; Gurskaya et al., FEBS Lett. 2001 Oct. 19;507(1): 16–20).
GFPs and RCFPs are stable proteins which, when they are produced accumulate in cells along with the protein of interest, allows easy detection of the movement and final degradation of that protein. The fusion of EGFP to amino acids 422 to 461 of MODC was shown to decreased the stability of EGFP(Li et al., J Biol Chem. 1998 Dec. 25;273(52):34970–5). The vector encoding for the resulting fusion protein is marketed by BD Biosciences Clontech as d2EGFP. Several point mutations in the MODC sequence yielded an EGFP protein with an half-life of 50 minutes compared to 26 hours for the unmodified EGFP (Corish et al, Protein Eng. 1999 December;12(12):1035–40). The latter fusion protein was called d1EGFP. Clontech researchers used d1EGFP to analyze the regulation of gene expression in live cells (Li et al., 1998).
Proteasome Sensors
Researchers have exploited the fact that d2EGFP is targeted for proteasomal degradation to monitor the activity of the proteasome in live cells. Variations in the activity of the proteasome result in variation in the fluorescent signal of cells expressing d2EGFP (Andreatta et al., Biotechniques. 2001 March;30(3):656–60; Nahreini et al., Cell Mol Neurobiol. 2001 October;21(5):509–21). Other fusion proteins between GFP and proteasome targeting motifs have also been successfully used for this purpose (Bence et al. Science. 2001 May 25;292(5521):1552–5; Dantuma et al. Nat Biotechnol. 2000 May;18(5):538–43). Fluorescent sensors of the activity of the proteasome are to date the most powerful tools to monitor the activity of the proteasome.
The Dantuma group (Nature biotechnology (2000) vol 18 p538–543) constructed a fusion of GFP to Ubiquitin using a standard peptide bond on the N-terminus. The ubiquitination occurs during translation, producing a peptide bond. The sensitivity achieved is —4 μM Z-L3-VS and 60 μM NP-LLG-VS at 15 hours using flow cytometry (FACS).
The Bence group out of Stanford (Science (2001) vol 292 p1552–1555) designed a proteasome sensor construct which is a fusion of GFP to an artificial peptide, CL1, identified in yeast. The CR1 sequences was inserted in 3′ of the reporter. The sensitivity achieved is 845 nM Lactacystin using flow cytometry.
The Andreatta group (Biotechniques (2001) vol 30 p656–660) has reported a construct designed from the fusion of GFP to a fragment of the Mouse Ornithine DeCarboxylase (MODC) protein. The sensitivity achieved is 2.5 uM Lactacystin and 2.5 uM MG132 at 20 hours using flow cytometry.
It would be an important advancement in the art to develop a sensor for proteasome activity with improved sensitivity, and other features to make available new, important areas of applications such as drug screening and analysis of the proteolytic systems of cells. Improvement of sensitivity of an order of magnitude or more over prior art systems would be of particular benefit.